Interleukins, interferons, colony stimulating factors and TNF.alpha. are examples of a group of diverse multi-functional proteins called cytokines. Cytokines are a class of secreted soluble proteins normally present in very low concentration in a variety of cells. Lymphoid, inflammatory hemopoietic and other cells such as connective tissue cells (e.g. fibroblasts, osteoblasts) secrete a variety of cytokines which regulate the immune, inflammatory, repair and acute phase responses by controlling cell proliferation, differentiation and effector functions. The effects of cytokines are mediated through binding to high affinity receptors on specific cell types. Collier et al., Trends in Pharmacol. Sci. 10: 427-431 (1989).
For example, the cytokine interleukin-1 (IL-1) is produced in cell types such as macrophages, synoviocytes, Iceratinocytes, chondrocytes and polymorphonuclear leukocytes. It is known to play a role in numerous conditions, in particular conditions accompanied by inflammation.
Several harmful effects are associated with increased IL-1. For example, in arthritis IL-1 stimulates synoviocytes associated with synovial hypertrophy. Further, IL-1 enhances cartilage matrix breakdown and inhibits cartilage repair by chondrocytes. This cytokine also induces bone resorption and thus may be involved in loss of bone density seen in rheumatoid arthritis. Weinblatt et al., Journal of Rheumatology 19:(Sup. 32):85-91(1992).
Excessive IL-1 production may cause fever, muscle wasting and drowsiness. For a review of the biological activities of IL-1, see Larrick, et al., Immunology Today 10:61-66 (1989). Thus, it is therapeutically desirable to inhibit the specific biological activity of IL-1.
One method of inhibiting IL-1 activity is by the use of systemic gene therapy. U.S. Pat. No. 5,766,585 discloses a method for treating rheumatoid arthritis inflammation and other autoimmune diseases in a mammal by administering a recombinant vector encoding an IL-1 antagonist.
TNF.alpha. is a cytokine that induces the production of IL-1. TNF.alpha. is a seventeen kDa peptide produced by activated macrophages as well as a wide variety of other cells during host immunological responses to microbial infections and neoplastic diseases. This cytokine is also recognized to be an important mediator of the inflammatory response. Beuller, et al., Ann. Rev. Immunol. 7: 625 (1989). It is therefore therapeutically desirable to inhibit the specific biological activity of TNF.alpha. as well as IL-1.
Another important cytokine is IL-10, a 35-40 kDa peptide produced by helper T-cells, B-cells, monocytes, macrophages and other cell types. In vitro, IL-10 has demonstrated immunosuppressive properties as evidenced by its ability to suppress cytokine production including IL-1 and TNF.alpha. (for a review, see Fiorentino et al., Journal of Immunology 147: 3815 (1991).
IL-10 also inhibits activation of other inflammatory cytokines, and therefore has potent anti-inflammatory activity. It is also known that IL-10 stimulates the proliferation of mast cells and thymocytes. Moore et al. "Interleukin-10," Annual Review in Immunology 11: 165-190 (1993).
It has been of recent interest to administer IL-10 in the treatment of certain conditions characterized by excessive IL-1 and TNF.alpha. production. Such diseases or conditions include loosening of prosthetic joint implants, inflammation, diabetes, cancer, graft versus host diseases, viral, fungal and bacterial infections, lipopolysaccharide endotoxin shock, diseases of depressed bone marrow function, thrombocytopenia, osteoporosis, spondyloarthropathies, Paget's disease, inflammatory bowel disease, arthritis, osteoarthritis, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and connective tissue diseases.
For example, purified IL-10 has been shown in vitro to suppress certain types of viral infections. U.S. Pat. No. 5,665,345 discloses a method for inhibiting replication of the human immunodeficiency virus, retro-viruses, and Kaposi sarcoma in human cells by administering IL-10.
IL-10 has also been suggested for use in the treatment of certain cancers. U.S. Pat. No. 5,570,190 discloses administering exogenous IL-10 to treat mammals suffering from acute myelogenous leukemia and acute lymphocytic leukemia. IL-10 is said to be administered either in the purified or recombinant form and is believed to inhibit the proliferation of acute leukemia blast cells. However, there is an enormous expense associated with preparing a purified or recombinant parenteral form of IL-10.
Similarly, IL-10 was shown to inhibit bone marrow metastasis in severe combined immunodeficient mice. Stearns et al., Invasion Metastasis 17(2):62-74 (1997).
The above conventional approaches to treating conditions characterized by excessive IL-1 and TNF.alpha. production have been limited to administering exogenous purified or recombinant IL-10 intravenously. Since IL-10 is a protein, it is difficult to infuse intravenously into a mammal because proteins often leach out of solution and bind to the plastic or glass used in intravenous administration sets. Also, proteins are often incompatible and precipitate when mixed with physiological solutions such as dextrose or saline. In addition, oral and topical routes are unavailable for IL-10 administration. The oral route is unavailable because protein is degraded in the gastrointestinal tract.
None of the above approaches suggests enhancing endogenous IL-10 production in mammals for prophylaxis and treatment of diseases or conditions using compounds that are approved for use in humans and are available in the oral, injectable and topical routes.
The compound, tetracycline, exhibits the following general structure: ##STR1## The numbering system of the ring nucleus is as follows: ##STR2##
Tetracycline as well as the 5-OH (Terramycin) and 7-Cl (Aureomycin) derivatives exist in nature, and are well known antibiotics. Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements of the structure must be retained. The modifications that may and may not be made to the basic tetracycline structure have been reviewed by Mitscher in The Chemistry of Tetracyclines, Chapter 6, Marcel Dekker, Publishers, New York (1978). According to Mitscher, the substituents at positions 5-9 of the tetracycline ring system may be modified without the complete loss of antibiotic properties. Changes to the basic ring system or replacement of the substituents at positions 1-4 and 10-12, however, generally lead to synthetic tetracyclines with substantially less or effectively no antimicrobial activity. An example of a chemically modified tetracyclines (hereinafter CMT) is 4-dedimethylaminotetracyline which is commonly considered to be a non-antimicrobial tetracycline.
The use of tetracycline antibiotics, while effective, may lead to undesirable side effects. For example, the long-term administration of antibiotic tetracyclines may reduce or eliminate healthy flora, such as intestinal flora, and may lead to the production of antibiotic resistant organisms or the overgrowth of opportunistic yeast and fungi. These side-effects of long-term tetracycline therapy can be particularly disadvantageous to patients with diabetes because these patients are particularly susceptible to infection and impaired wound healing which might, at some future time, require antibiotic therapy to combat infection.
In addition to their antibiotic properties, tetracyclines have been described as having a number of other uses. For example, tetracyclines are also known to inhibit the activity of collagen destructive enzymes such as mammalian collagenase, gelatinase, macrophage elastase and bacterial collagenase. Golub et al., J. Periodont. Res. 20:12-23 (1985); Golub e t al. Crit. Revs. Oral Biol Med. 2: 297-322 (1991);U.S. Pat. Nos. 4,666,897; 4,704,383; 4,935,411; 4,935,412. In addition, tetracyclines have been known to inhibit wasting and protein degradation in mammalian skeletal muscle, U.S. Pat. No. 5,045,538.
Furthermore, tetracyclines have been shown to enhance bone protein synthesis in U.S. Pat. No. Re. 34,656, and to reduce bone resorption in organ culture in U.S. Pat. No. 4,704,383.
Similarly, U.S. Pat. No. 5,532,227 to Golub et al, discloses that tetracyclines can ameliorate the excessive glycosylation of proteins. In particular, tetracyclines inhibit the excessive collagen cross linking which results from excessive glycosylation of collagen in diabetes.
These properties cause the tetracyclines to be useful in treating a number of diseases. For example, there have been a number of suggestions that tetracyclines, including non-antimicrobial tetracyclines, are effective in treating arthritis. See, for example, Greenwald et al., "Tetracyclines Suppress Metalloproteinase Activity in Adjuvant Arthritis and, in Combination with Flurbiprofen, Ameliorate Bone Damage," Journal of Rheumatology 19:927-938(1992); Greenwald et al., "Treatment of Destructive Arthritic Disorders with MMP Inhibitors: Potential Role of Tetracyclines in, Inhibition of Matrix Metalloproteinases:Therapeutic Potential," Annals of the New York Academy of Sciences 732: 181-198 (1994); Kloppenburg et al., "Minocycline in Active Rheumatoid Arthritis," Arthritis Rheum 37:629-636(1994); Ryan et al., "Potential of Tetracycline to Modify Cartilage Breakdown in Osteoarthritis," Current Opinion in Rheumatology 8: 238-247(1996); O'Dell et al., "Treatment of Early Rheumatoid Arthritis with Minocycline or Placebo," Arthritis Rheum 40:842-848(1997).
Tetracyclines have also been suggested for use in treating skin diseases. For example, White et al., Lancet, Apr. 29, p. 966 (1989) report that the tetracycline minocycline is effective in treating dystrophic epidermolysis bullosa, which is a life-threatening skin condition believed to be related to excess collagenase.
The effectiveness of tetracycline in skin disorders has also been studied by Elewski et al., Journal of the American Academy of Dermatology 8:807-812 (1983). Elewski et al. disclosed that tetracycline antibiotics may have anti-inflammatory activity in skin diseases.
Similarly, Plewig et al., Journal of Investigative Dermatology 65:532 (1975), disclose experiments designed to test the hypothesis that antimicrobials are effective in treating inflammatory dermatoses. The experiments of Plewig et al. establish that tetracyclines have anti-inflammatory properties in treating pustules induced by potassium iodide patches.
The use of tetracyclines in combination with non-steroidal anti-inflammatory agents has been studied in the treatment of inflammatory skin disorders caused by acne vulgaris. Wong et al., Journal of American Academy of Dermatology 1: 1076-1081 (1984), studied the combination of tetracycline and ibuprofen and found that tetracycline was an effective agent against acne vulgaris while ibuprofen was useful in reducing the resulting inflammation by inhibition of cycloxygenase. Funt et al., Journal of the American Academy of Dermatology 13: 524-525 (1985), disclosed similar results by combining antimicrobial doses of minocycline with ibuprofen.
An antimicrobial tetracycline derivative, doxycycline, has been used to inhibit nitrate production. D'Agostiiio et al., Journal of Infectious Diseases: 177:489-92 (1998), disclose experiments where doxycycline, administered to mice injected with bacterial lipopolysaccharide (hereinafter LPS), exerted a protective effect by inhibiting nitrate production by an IL-10 independent mechanism. Experiments carried out in vitro also showed that doxycycline inhibited nitric oxide synthesis by LPS activated macrophages without enhancing endogenous IL-10 release. These data are contrary to the results of the present invention.
Based on the foregoing, tetracyclines have been found to be effective in different treatments. However, there has been no suggestion whatsoever that tetracyclines can be used to enhance endogenous production of IL-10.
Accordingly, it is one of the advantages of the present invention to overcome the above limitations of administering exogenous IL-10 and provide a method for enhancing endogenous IL-10 production in mammalian cells. Other advantages will readily present themselves to the skilled practitioner.